Estradiol (E2) binds to Estrogen Receptor alpha (ERα) activation functions (AF-1 and -2) and regulates gene transcription. A fraction of ERa is targeted to plasma membrane and elicits membrane-initiated steroid signalling (MISS) but its physiological role has never been directly investigated in vivo. We thus generated a mouse mutated for the palmitoylation site (C451A-ERα) by homologous recombination, leading to an abrogation of membrane localization of ERα. Surprisingly, this single mutation leads to total female infertility with abnormal ovaries lacking corpus luteum and hence low progesterone production. However, E2 proliferative action was completely preserved in the uterus of C451A-ERa mutant mice and endometrial epithelial proliferation was similar to wild-type. On the opposite, E2 vascular effects, such as rapid dilatation, acceleration of endothelial healing and endothelial NO synthase phosphorylation were abrogated in arteries of C451A-ERa mice. In striking contrast, in mice inactivated for genomic effects (ERa-AF2°), acceleration of re-endothelialization using membrane-selective activator estrogen-dendrimer conjugate (EDC) was preserved, demonstrating the integrity of the MISS actions in ERa-AF2°. Using a large scale analysis of uterine gene expression, almost all uterine E2-dependent gene expression was abrogated in ERa-AF2°, whereas in C451A mice, gene regulation was essentially similar to wild-type, revealing that this uterine transcriptional response depends primarily on genomic/nuclear functions of ERα. These models provide evidence for the first time of the physiological role of MISS effects of ERα in vivo and delineate tissue specific roles of membrane and nuclear actions of ERα.

Significance The in vivo roles of plasma membrane-associated estrogen receptor (ER)α, including cross-talk with nuclear ERα, are poorly understood. We created a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function. A complementary mouse lacking the ERα activation function AF-2 (ERα-AF2 0 ) provided selective loss of function of nuclear ERα actions. Physiologic studies revealed critical requirements for membrane receptors in ovarian function and thereby in fertility, and in vascular physiology. In contrast, nuclear ERα actions mediate uterine responses to estrogen and genome-wide analysis indicates that membrane-to-nuclear receptor cross-talk in vivo is quite modest in uterus. These findings demonstrate for the first time critical tissue-specific roles for membrane versus nuclear actions of a steroid hormone receptor in vivo.

High estradiol levels in late puberty induce growth plate closure and thereby cessation of growth in humans. In mice, the growth plates do not fuse after sexual maturation, but old mice display reduced longitudinal bone growth and high-dose estradiol treatment induces growth plate closure. Estrogen receptor (ER)-α stimulates gene transcription via two activation functions (AFs), AF-1 and AF-2. To evaluate the role of ERα and its AF-1 for age-dependent reduction in longitudinal bone growth and growth plate closure, female mice with inactivation of ERα (ERα(-/-)) or ERαAF-1 (ERαAF-1(0)) were evaluated. Old (16- to 19-mo-old) female ERα(-/-) mice showed continued substantial longitudinal bone growth, resulting in longer bones (tibia: +8.3%, P < 0.01) associated with increased growth plate height (+18%, P < 0.05) compared with wild-type (WT) mice. In contrast, the longitudinal bone growth ceased in old ERαAF-1(0) mice (tibia: -4.9%, P < 0.01). Importantly, the proximal tibial growth plates were closed in all old ERαAF-1(0) mice while they were open in all WT mice. Growth plate closure was associated with a significantly altered balance between chondrocyte proliferation and apoptosis in the growth plate. In conclusion, old female ERα(-/-) mice display a prolonged and enhanced longitudinal bone growth associated with increased growth plate height, resembling the growth phenotype of patients with inactivating mutations in ERα or aromatase. In contrast, ERαAF-1 deletion results in a hyperactive ERα, altering the chondrocyte proliferation/apoptosis balance, leading to growth plate closure. This suggests that growth plate closure is induced by functions of ERα that do not require AF-1 and that ERαAF-1 opposes growth plate closure.

Alkylated polycyclic aromatic hydrocarbons (APAHs) are the main components of polycyclic aromatic hydrocarbons (PAHs) in petroleum-contaminated waters. In our study, three kinds of green microalgae (Pseudokirchneriella subcapitata, Chlorella vulgaris and Scenedesmus obliquus) were shown to degrade six kinds of methylphenanthrenes (1-methylphenanthren, 2-methylphenanthrenem, 3-nmmethylphenanthrene, 4-methylphenanthren, 9-methylphenanthrene and 3,6-dimethylphenanthrene) with different degrading abilities. Among the six methylphenanthrenes, 99.8% of 1-methylphenanthrene (1-MP) was removed and 75.6% of 3,6-dimethylphenanthrene (3,6-DMP) was removed by P. subcapitata after 7 days of incubation. The metabolites of 1-MP and 3,6-DMP were identified by gas chromatograph-mass spectrometer (GC-MS). Six metabolites of 1-MP and one metabolite of 3,6-DMP were found, they were all monohydroxylated methylphenanthrenes. The –OH group was added to either methyl-group or benzene ring through the monooxygenase system, and the methyl-group attack was the main pathway. This research increases our knowledge of the degrading ability of APAHs by green microalgae and offers information for the bioremediation of APAHs. Quantum chemical calculation was conducted to elucidate the biodegradation metabolites of methylphenanthrene by green microalgae, which is a helpful tool in the bioremediation of environmental pollution.

Les œstrogènes préviennent l'accumulation de masse grasse et l'installation d'une insulinorésistance via leur récepteur α (ERα). Ce récepteur nucléaire est connu pour réguler la transcription de nombreux gènes grâce à deux fonctions de transactivation, AF-1 et AF-2, et nous avons récemment montré que, en activant le récepteur indépendamment de la fonction AF-1, il est possible de conserver l'intégralité des effets protecteurs des œstrogènes sur le métabolisme glucidique et la paroi vasculaire, sans effet délétère sur l'utérus. Pour déterminer l'implication de la fonction AF-2 dans l'action bénéfique des œstrogènes sur la composition corporelle, la sensibilité à l'insuline et l'homéostasie glucidique, des souris déficientes en ERα (ERα−/−) ou spécifiquement en AF-2 (AF-2°), ont été étudiées en conditions de régime standard ou hyperlipidique (mâles et femelles, n = 8–10 par sexe et par génotype). Par comparaison à leurs contrôles sauvages, les souris ERα−/− et AF-2° sont caractérisées par une prise de poids accélérée avec accumulation massive de tissu adipeux, une insulinorésistance et une intolérance au glucose. Pour s'affranchir de l'effet potentiel des stéroïdes sexuels endogènes, des souris femelles ovariectomisées ont ensuite reçu une administration chronique de 17β- estradiol à doses physiologiques. Comme attendu, ce traitement prévient l'excès d'adiposité, ainsi que l'altération de la sensibilité à l'insuline (clamps hyperinsulinémiques, voie de signalisation de l'insuline dans les tissus périphériques) et de la tolérance au glucose chez les souris sauvages, mais ces effets sont totalement abolis chez les souris ERα−/− et AF-2°. Comme précédemment démontré concernant leurs effets vasculoprotecteurs, l'action bénéfique des œstrogènes sur l'adiposité, la sensibilité à l'insuline et la tolérance au glucose s'avère indépendante de la fonction AF-1, mais dépendante de la fonction AF-2 du ERα. Ces résultats ouvrent la voie à une modulation sélective du récepteur activant spécifiquement AF-2 dans une perspective de prévention cardiovasculaire et métabolique.

Lignans are plant compounds metabolized in the mammalian gut to produce the estrogenic enterolignans, enterodiol (ED) and enterolactone (EL). Because estrogens have been linked to breast cancer etiology, enterolignans could affect breast cancer risk, but to our knowledge, the mechanisms by which they exert their estrogenic and/or anti-estrogenic effects in humans are still unclear. To better understand how estrogenic compounds from the food, such as the enterolignans, might influence breast cancer progression and their mechanisms to interfere with human estrogen receptor (ER) signalling in hormone-dependant diseases, we examined and compared the ability of ED, EL and 17β-estradiol (E2) to induce the transactivation of ERα and ERβ, to modulate ERα target genes, to exert either growth stimulatory or anti-proliferative effects and finally to modulate MCF-7 cell migration by acting on matrix metalloproteases (MMP)-2 and -9, at concentrations that are achievable through a lignan-rich diet. This study indicates that enterolignans show distinct properties for transactivation of ERα and ERβ. ED, as E2, induces ERα transcriptional activation through transactivation functions AF-1 and AF-2, while EL is less efficient in inducing AF-1, acting predominantly through AF-2. Furthermore, ED and EL modulate ERα mRNA and protein contents as well as MCF-7 cell proliferation and secreted MMP activities in a different way. Enterolignans are compounds of wide interest nowadays and our results help to unveil their mechanisms of action on ER, emphasizing the fact that the dietary load in lignans could be of importance in the balance between being risk or chemopreventive factors for breast cancer and women's health.

The bone-sparing effect of estrogen is primarily mediated via estrogen receptor-α (ERα), which stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal and AF-2 in the ligand binding domain. To evaluate the role of ERα AF-1 and ERα AF-2 for the effects of estrogen in bone in vivo, we analyzed mouse models lacking the entire ERα protein (ERα−/−), ERα AF-1 (ERαAF-10), or ERα AF-2 (ERαAF-20). Estradiol (E2) treatment increased the amount of both trabecular and cortical bone in ovariectomized (OVX) WT mice. Neither the trabecular nor the cortical bone responded to E2 treatment in OVX ERα−/− or OVX ERαAF-20 mice. OVX ERαAF-10 mice displayed a normal E2 response in cortical bone but no E2 response in trabecular bone. Although E2 treatment increased the uterine and liver weights and reduced the thymus weight in OVX WT mice, no effect was seen on these parameters in OVX ERα−/− or OVX ERαAF-20 mice. The effect of E2 in OVX ERαAF-10 mice was tissue-dependent, with no or weak E2 response on thymus and uterine weights but a normal response on liver weight. In conclusion, ERα AF-2 is required for the estrogenic effects on all parameters evaluated, whereas the role of ERα AF-1 is tissue-specific, with a crucial role in trabecular bone and uterus but not cortical bone. Selective ER modulators stimulating ERα with minimal activation of ERα AF-1 could retain beneficial actions in cortical bone, constituting 80% of the skeleton, while minimizing effects on reproductive organs.

Les œstrogènes préviennent l'accumulation de masse grasse et la survenue d'un diabète de type 2 via leur récepteur α(ERα), mais leurs effets indésirables limitent leur intérêt thérapeutique. ER_régule la transcription de nombreux gènes grâce à ses deux fonctions de transactivation, AF-1 et AF-2, et nous avons montré que, en activant le récepteur indépendamment de la fonction AF-1, il est possible de conserver l'intégralité des actions vasculoprotectrices des oestrogènes sans effet délétère sur l'utérus. Pour étudier l'implication des fonctions de transactivation de ER_dans l'action bénéfique des oestrogènes sur la composition corporelle et l'homéostasie glucidique, des souris déficientes en ERα(ERα-/-), en AF-1 (AF-1-/-) ou AF-2 (AF-2-/-), ont été suivies jusqu'à l'âge de 7 mois (régime standard, mâles et femelles, n = 8–10 par sexe et par génotype). Par comparaison à leurs contrôles sauvages, les souris ERα-/- ont été caractérisées par une prise de poids accélérée avec accumulation massive de tissu adipeux, une insulinorésistance et une intolérance au glucose. Les souris AF-2-/- ont développé un profil phénotypique et métabolique identique. En revanche, nous n'avons pas observé d'accélération du gain pondéral et du développement de la masse grasse chez les animaux AF-1-/-. De plus, la tolérance au glucose était conservée chez les mâles, et altérée uniquement tardivement chez les femelles AF-1-/-. De façon concordante, l'effet protecteur de l'administration chronique de 17β-estradiol vis-à-vis de l'accumulation de tissu adipeux chez des femelles ovariectomisées était aboli chez les souris ERα-/- et AF-2-/-, mais totalement conservé chez les souris AF-1-/-. Comme pour leurs effets vasculoprotecteurs, l'action favorable des oestrogènes sur la masse grasse et la tolérance au glucose est en grande partie indépendante de la fonction AF-1 du ERα. Ces résultats ouvrent la voie à une modulation sélective du récepteur n'activant pas AF-1 dans une perspective de prévention cardiovasculaire et métabolique.

Post-menopausal women have an increased risk of developing a number of degenerative pathological conditions, linked by the common theme of excessive inflammation. Systemic estrogen replacement (in the form of hormone replacement therapy) is able to accelerate healing of acute cutaneous wounds in elderly females, linked to its potent antiinflammatory activity. However, in contrast to many other age-associated pathologies, the detailed mechanisms through which estrogen modulates skin repair, particularly the cell type–specific role of the two estrogen receptors, ERα and ERβ, has yet to be determined. Here, we use pharmacological activation and genetic deletion to investigate the role of both ERα and ERβ in cutaneous tissue repair. Unexpectedly, we report that exogenous estrogen replacement to ovariectomised mice in the absence of ERβ actually delayed wound healing. Moreover, healing in epidermal-specific ERβ null mice (K14-cre/ERβL2/L2) largely resembled that in global ERβ null mice. Thus, the beneficial effects of estrogen on skin wound healing are mediated by epidermal ERβ, in marked contrast to most other tissues in the body where ERα is predominant. Surprisingly, agonists to both ERα and ERβ are potently antiinflammatory during skin repair, indicating clear uncoupling of inflammation and overall efficiency of repair. Thus, estrogen-mediated antiinflammatory activity is not the principal factor in accelerated wound healing.

Full-length 66-kDa estrogen receptorα (ERα) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46-kDa ERα isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. Previous studies in cultured endothelial cells showed that the N-terminal A/B domain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ERαAF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ERα A/B domain in the mouse. In these ERαAF-1zero mice, both basal endothelial NO production and reendothelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18-weekold ERαAF-1+/+LDLR-/- (low-density lipoprotein receptor) and ERαAF-1zeroLDLR-/- mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the ic tree of 8-month-old ovariectomized or intact female mice revealed that ERαAF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ERαAF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, selective ER modulators stimulating ERα with minimal activation of ERαAF-1 could retain beneficial vascular actions, while minimizing the sexual effects.

Learning motor skills is critical for motor abilities such as driving a car or playing piano. The speed at which we learn those skills is subject to many factors. Yet, it is not known to what extent gonadal hormones can affect the achievement of accurate movements in time and space. Here we demonstrate via different lines of evidence that estradiol promotes plasticity in the cerebellar cortex underlying motor learning. First, we show that estradiol enhances induction of long-term potentiation at the parallel fiber to Purkinje cell synapse, whereas it does not affect long-term depression; second, we show that estradiol activation of estrogen receptor β receptors in Purkinje cells significantly improves gain-decrease adaptation of the vestibulo-ocular reflex, whereas it does not affect general eye movement performance; and third, we show that estradiol increases the density of parallel fiber to Purkinje cell synapses, whereas it does not affect the density of climbing fiber synapses. We conclude that estradiol can improve motor skills by potentiating cerebellar plasticity and synapse formation. These processes may be advantageous during periods of high estradiol levels of the estrous cycle or pregnancy.

Recent studies have revealed that hundreds of genes in the uterus are activated by estrogen. Their expression profiles differ over time and doses and it is not clear whether all these genes are directly regulated by estrogen via the estrogen receptor. To select the genes that may be regulated by estrogen, we treated mice with several doses of estrogen and searched for those genes whose dose-response expression pattern mirrored the uterine growth pattern. Among those genes, we found that the dose-dependent expression of the adrenomedullin (ADM) gene correlated well with the uterotrophic effect of estrogen. ADM expression is induced early after estrogen administration and is restricted to the endometrial stroma. The spatiotemporal gene expression pattern of ADM was similar to that of receptor-modifying protein 3 (RAMP3). RAMP3 is known to modify calcitonin gene-related receptor (CRLR) so that it can then serve as an ADM receptor. Chromatin immunoprecipitation assays indicated that the estrogen receptor binds directly to the ADM promoter region and RAMP3 intron after estrogen administration. It was also shown that neither the ADM nor RAMP3 gene could be activated in estrogen receptor-alpha-null mouse. Although uterine ADM expression has been reported to occur in the myometrium, our observations indicate that estrogen-induced ADM is also expressed in the uterine stroma and that such variable, spatiotemporally regulated ADM expression contributes to a wider range of biological effects than previously expected.